1. Field of Invention
This invention relates to regulating Anthrolysin O expression in Bacillus anthracis and Bacillus cereus and Bacillus thuringiensis. 
2. Description of Related Art
Bacillus anthracis is the causative agent of anthrax, an often fatal zoonosis. B. anthracis is a large, non-motile, Gram-positive rod shape bacteria. When B. anthracis encounter stress or nutrient limitation it produces spores. Spores are resistant to a wide range of physical and chemical agents and can survive in the soil for decades. The hardiness of the spores together with the ease of producing them make that B. anthracis was developed as a bio-weapon. The bio-terror letter attacks of 2001 have stressed the need of a better understanding of the disease to prevent and cure possible future attacks.
For more than fifty years only two virulence factors have been known and intensely studied: the poly-D-glutamyl capsule which is encoded by virulence plasmid pX02, and. anthrax toxin, encoded by pXO1. Anthrax toxin is made of three peptides: lethal factor (LF), edema factor (EF) and protective antigen (PA). LF or EF were combined with P A to make two different A/B7 toxins. The seven P A subunits form a pore that transports the enzymatic toxin part (LF or EF) into the target cells. LF is zinc metalloprotease that cleaves the MAPK kinase, an important enzyme in intracellular signal transduction. EF is a calcium and calmodulin-dependent adenylate cyclase that increases the intracellular amount of cAMP thus disrupting the flow of ions and water and causing edema.
These virulence factors account for the some of the pathology, morbidity and mortality observed with B. anthracis and have been extensively studied. In addition to those well studied virulence factors, B. anthracis genome analysis has shown a whole array of other putative virulence factors: phospholipases, enterotoxins, hemolysins, and a cholesterol dependant cytolysin (CDC) (Read T D et al. The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature. 2003 May 1; 423(6935):81-6.). The roles of these virulence factors remain to be elucidated. Inventors have focused on the study B. anthracis cholesterol-dependent cytolysin (CDC) named anthrolysin O (ALO) (Shannon J G, Ross C L, Koehler T M, Rest R F. Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin. Infect Immun. 2003 June; 71(6):3183-9; Mosser E M, Rest R F. The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages. BMC Microbiol. 2006 Jun. 21; 6:56; Park J M, Ng V H, Maeda S, Rest R F, Karin M. Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists. J Exp Med. 2004 Dec. 20; 200(12): 1647-55, all of which are incorporated by reference herein in their entireties). ALO has been described in Patent Application Publication US2006/0089305A1 which is incorporated herein in its entirety. CDCs are a family of pore-forming cytolysins that are proven virulence factors in more than 20 phylogenically unrelated Gram-positive pathogenic bacteria among which are Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, and Clostridium perfringens. The formation of pores by most CDCs are dependent upon the presence of cholesterol in the membranes. CDCs are secreted as soluble monomeric proteins that bind to membranes and oligomerizes on the surface. The oligomers then insert into the membrane forming a pore. The CDC pore is made of 35 to 50 monomers and has a diameter of 25 to 30 nm.
Bacillus cereus and Bacillus thuringiensis, two species closely related to B. anthracis express CDCs, cereolysin O (CLO) and thuringiolysin O (TLO), respectively. In these organisms the expression of CDCs is controlled by the pleiotropic transcriptional activator PlcR. The activation of the PlcR regulon occurs at the onset of stationary phase by a quorum sensing mechanism and is triggered by a small peptide PapR (Ross C L, Koehler T M. plcR papR-independent expression of anthrolysin O by Bacillus anthracis. J. Bacteriol. 2006 November; 188(22):7823-9, incorporated by reference herein in its entirety). In B. anthracis, PlcR is inactive due to a nonsense mutation in the plcR gene. Therefore, it was believed that the PlcR regulon was silent in B. anthracis and, in particular, it was perceived that it produced no CDC and was therefore non-hemolytic. To the contrary, inventors have discovered contrary evidence that the B. anthracis CDC, ALO, is regulated by a mechanism different from B. cereus and B. thuringiensis. 
All references cited herein are incorporated herein by reference in their entireties.